The overall goal of this proposal is to elucidate at the molecular level signaling pathways from the insulin receptor. Many of these pathways begin with tyrosine phosphorylation of the insulin receptor substrates (IRS's) by the activated insulin receptor. The phosphorylated IRS's then function as docking/effector proteins for SH2 domain-containing signaling proteins. The IRS's thus have a key role in signaling from the insulin receptor. Until recently, only two members of the IRS family, IRS-1 and IRS-2, were known. In the past three years we have discovered and begun the characterization of two additional members of the IRS family, IRS-3 and IRS-4. IRS-3 and IRS-4 are most abundant in adipocytes and human embryonic kidney 293 cells, respectively; and we have determined the SH2 domain proteins associated with them in these cell types. In addition, we have generated mice with targeted disruption of the IRS-3 and IRS-4 genes; initial analyses of these knockout (ko) mice have not revealed abnormalities in glucose homeostasis or otherwise. One specific aim of this proposal is to determine the physiological roles of IRS-3 and IRS-4. This will be achieved by further characterization of the IRS-3 and IRS-4 ko mice. Investigation of the IRS-3 ko mice will focus on the effects of IRS-3 deletion on insulin signaling in adipocytes. In addition, in order to detect overlapping and/or compensatory roles for the IRS's, mice lacking two IRS's will be generated and characterized. The following three double ko's will be examined in our laboratory: IRS-1,4; IRS-2 and either 3 or 4; and IRS-3,4. The other double ko's will be examined in the laboratories of collaborators. The second specific aim of this proposal is to discover and characterize novel proteins that are components of signaling pathways involving IRS-3 or IRS-4. This will be done by searching for proteins that interact with IRS-3 and IRS-4. Already we have found a group of eight proteins that are associated with IRS-4 isolated by immunoprecipitation. These will be characterized. In addition, proteins associated with IRS-3 will be found by screening expression libraries with 32P-labeled IRS-3 and by screening with various domains of IRS-3 as bait in a yeast two-hybrid system.